Most phosphorylated cellular proteins are phosphorylated on threonine and serine residues; however, an important class of proteins is phosphorylated on tyrosine residues. Tyrosine kinase activity has been found in a number of membrane bound ligand receptors, such as the epidermal growth factor (EGF) receptor and the insulin receptor. Furthermore, a number of oncogenes encode proteins that have tyrosine kinase activity, these oncogenes include v-erb, v-fps, v-abl, and v-src. The phosphorylation of specific proteins by tyrosine kinases is believed to have important physiological consequences for the cell.
Identified substrates for tyrosine kinases include the GAP protein. Guanosine triphosphatase (GTPase) activator protein, referred to as GAP, stimulates the weak intrinsic GTPase activity of normal ras. GAP acts on normal ras p21 and converts it to a ras p21.GDP complex. In contrast, oncogenic forms of ras p21 are not sensitive to GAP, and persist as ras p21.GTP complexes. It is believed that GAP may attenuate signaling by normal ras p21.GTP. Some studies suggest that GAP may itself be the effector through which ras p21.GTP transmits a mitogenic signal to the cell. Mutagenesis of the GAP interaction domain on oncogenic forms of ras p21 blocks signaling. McCormick, F., 1989, Cell, 56: 5; Adari. H., et al., 1988 Nature, 332: 548. Injection of a truncated form of ras p21, that has an increased affinity for GAP into Xenopus oocyte blocked some effects of oncogenically activated ras, and excess GAP overcome this inhibition, Gibbs, J. B., et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 6630. Krev, a protein that blocks the transforming effects of oncogenic ras mutants on cells is very similar to ras in the GAP-binding domain and may act by competing with ras for binding to GAP. Kitayama, H., et al. 1989, Cell, 56: 77.
Experiments have shown that GAP is spatially associated with other proteins that are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. Immunoprecipitation of proteins in transformed mouse fibroblasts with GAP specific antiserum coprecipitates several proteins. When GAP immunoprecipitates are separated by SDS.sub.-- PAGE, western blotted to nitrocellulose, and subsequently probed with anti-phosphotyrosine specific antibodies, bands with relative molecular weight of 62,000 and 190,000 daltons, p62 and p190, respectively) are revealed; Ellis et al., Nature 343: 377-381 (1990). GAP, p62 and p190, have been shown to be tyrosine phosphorylated in cells transformed by v-scr, v-abl, and h-ras, as well as being tyrosine phosphorylated in response to stimulation of cells by EGF (Ellis, ibid.). The p62 protein has also been shown to specifically associate with the SH2 domain of GAP by means of experiments in which bacterially produced SH2 domains of GAP were reconstituted in vitro with p62, Moran et al, Function and Evolution of Ras Proteins, Cold Spring Harbor, N.Y. (1990). Furthermore, it has also been found that activated platelet derived growth factor receptor also binds to GAP, indicating that GAP may be involved in mediating PDGF's biological actions, Kazlanskas, Science, 247: 1578 (1990).